Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.     

Synthesis and biological activity of brassinolide and its 22β,23β-isomer: Novel plant growth-promoting steroids

Brassinolide (2α,3α,22α, 23α-tetrahydroxy-24α-methyl-B-homo-7-oxa-5α-cholestan-6-one), a novel plant growth-promoting steroid isolated from rape pollen, and its hitherto unknown 22β, 23β-isomer were synthesized from a C-24 epimeric 60:40 mixture of 22-dehydrocampesterol (24α-methyl) and brassicasterol (24β-methyl) from oysters. The method of synthesis favored the formation of the 22β, 23β-isomer by better than 4:1. Comparative plant growth-promoting capabilities of brassinolide, both natural and synthetic, and its three side chain $\text{cis}$-glycolic isomers in the bean second internode bioassay showed that the natural and synthetic brassinolides were equally active and caused splitting of the internode at the 0.1 μg level. The least active was the 22β,23β-isomer of brassinolide. The isomers with the 22α, 23α and 24β, and the 22β, 23β and 24β configurations were highly active and were required at about 10 times the concentration of brassinolide to cause the same physiological response. In the bean first internode bioassay, an auxin-induced growth test system which employs isolated bean plant segments, the isomer with 22β, 23β and 24β configuration caused a greater response than brassinolide. Two of the four tetrahydroxy ketones obtained in the synthesis of the isomers were also active in both assays.     

21 Ribosomal evidence suggests that archaea evolved after fungi

We are exploring the potential to trace species evolution with the ribosomal proteins (RibPs) present in bacterial, eukaryotic, and archaeal ribosomes and to compare the independent trees for consistency. The complete genomes of over 8400 bacteria, eukaryota, and archaea are presently in the SwissPro/TrEMBL (SPT) database. A search of SPT using a vector designed with ScanProsite formats (V1) finds and aligns 8405 sequences (5312 bacterial, 2905 eukaryotic, and 169 archaeal) that are homologous with bone fide bacterial S19 ribosomal proteins(S19s). When the 8405 sequences are perfectly aligned, 15 residues are conserved at 90% identity and 40 are conserved at 70% identity. We are not aware of any previous publication reporting sequence alignment of 8400 members of any single family including all bacteria, eukaryota and archaea, for which complete genomes have been published.A Pro and a Gly separated by 11 residues are 100% conserved in the 8405 S19s. In the position immediately before the fully conserved Gly, two residues (Asp and Asn) are present in 98.3% of the 8405 sequences. The Asp residue is found almost exclusively in 2190 gram-positive bacteria. The Asn residue is found in 3065 gram-negative bacteria, 123 Archaea, 1939 eukaryotes, and 64 specific species of gram-positive bacteria. There is biochemical evidence for the existence of distinct mitochondrial, chloroplast, and cytosolic ribosomes and reports that plants have all three forms and mammals only two. Reliable data concerning how individual ribosomal proteins differ in different types of ribosomes are meager. Examination of the eukaryotic S19s reveals the existence of three distinct types. Two of the distinctly different types are found in most fungi, three of the types are found in some viridiplante, and only one type is found in metazoa and archaea. We demonstrate the sequence homology between the mitochondrial form found in fungi and plants and the S19 proteins of alpha proteobacteria; between the chloroplast S19s and the S19s of cyanobacteria; and among the cytosolic S19s found only in fungi, metazoa, archaea, and in some viridiplantae. Our findings suggest that most archaeal species appeared after a gene duplication event in fungi that correlates with the origin of the cytosolic ribosome.     

STRUCTURE,LIFE HISTORY AND SYSTEMATICS OF RHOICOSPHENIA (BACILLARIOPHYTA). V. INITIAL CELL AND SIZE REDUCTION IN RH. CURVATA AND A DESCRIPTION OF THE RHOICOSPHENIACEAE FAM. NOV.1

The initial epivalve of Rhoicosphenia curvata (Kütz.) Grun. differs from vegetative valves in having a strongly arched section, a wide hyaline marginal strip, no pseudosepta, an unthickened margin, and a terminal raphe fissure at the head pole. The initial epivalve is of the D type, with short raphe fissures. The epicingulum consists of three bands as usual, but they are narrower and more delicate than those of vegetative cells. The initial hypovalve and hypocingulum are similar in every way to those of vegetative cells, except for the rounded section of the hypovalve. During size reduction the almost isopolar outline of the initial valves and their immediate descendants gives way to an increasingly strong heteropolarity, and this is accompanied by changes in the relative lengths of the raphe slits and the shape of the central area. Different populations have different gametangium and initial cell sizes, suggesting the presence of races within the species. The structure of the initial cell indicates that Rhoicosphenia is less closely related to the monoraphid genera than to the gomphocymbelloid genera, confirming conclusions reached from studies of the vegetative cell and auxospore formation. Rhoicosphenia should therefore be separated into a new family, the Rhoicospheniaceae, which is described.     

Evolution of pigment cell regression in the cavefish Astyanax: a late step in melanogenesis

Pigmentation and eyes are often lost in cave-adapted animals. Although the mechanisms of eye degeneration are beginning to be understood, little is known about the evolutionary and developmental processes involved in pigment cell regression. In teleost embryos, a population of neural crest cells migrates into the body wall and differentiates into melanophores, xanthophores, and iridophores. All three pigment cell types are present in the eyed surface-dwelling form (surface fish) of the teleost Astyanax mexicanus. However, melanophores are absent or substantially reduced in number in various derived populations of the conspecific blind cave-dwelling form (cavefish). We show here that tyrosinase-positive melanoblasts are present in cavefish. DiI labeling revealed a population of trunk neural crest cells in cavefish embryos that migrate to locations normally occupied by differentiated melanophores. We also discovered a cell population in cavefish embryos and adults resembling melanoblasts in several features, including the ability to synthesize melanin when supplied with the tyrosinase substrate l-dopa. DiI-tyrosinase double-labeling and neural keel explant experiments showed that the tyrosinase-positive cells are derived from the neural crest. The number of melanoblasts varies in different adult cavefish populations relative to the extent of melanophore reduction. Although cavefish melanoblasts can synthesize melanin from exogenous l-dopa, they are unable to convert exogenous l-tyrosine to l-dopa and melanin. We conclude that pigment cell regression in cavefish is mediated by an evolutionary change late in melanogenesis that may involve an impediment in the ability to convert l-tyrosine to l-dopa and melanin.     

Through the Eyes of Children: Perceptions of Environmental Change in Tropical Forests

This study seeks to understand children''s perceptions of their present and future environments in the highly biodiverse and rapidly changing landscapes of Kalimantan, Indonesian Borneo. We analyzed drawings by children (target age 10–15 years) from 22 villages, which show how children perceive the present conditions of forests and wildlife surrounding their villages and how they expect conditions to change over the next 15 years. Analyses of picture elements and their relationships to current landscape variables indicate that children have a sophisticated understanding of their environment and how different environmental factors interact, either positively or negatively. Children appear to have landscape-dependent environmental perceptions, showing awareness of past environmental conditions and many aspects of recent trends, and translating these into predictions for future environmental conditions. The further removed their present landscape is from the originally forested one, the more environmental change they expect in the future, particularly declines in forest cover, rivers, animal diversity and increases in temperature and natural disasters. This suggests that loss of past perceptions and associated “shifting environmental baselines” do not feature strongly among children on Borneo, at least not for the perceptions we investigated here. Our findings that children have negative expectations of their future environmental conditions have important political implications. More than other generations, children have a stake in ensuring that future environmental conditions support their long-term well-being. Understanding what drives environmental views among children, and how they consider trade-offs between economic development and social and environmental change, should inform optimal policies on land use. Our study illuminates part of the complex interplay between perceptions of land cover and land use change. Capturing the views of children through artistic expressions provides a potentially powerful tool to influence public and political opinions, as well as a valuable approach for developing localized education and nature conservation programs.     

Selection for High Oridonin Yield in the Chinese Medicinal Plant Isodon (Lamiaceae) Using a Combined Phylogenetics and Population Genetics Approach

Oridonin is a diterpenoid with anti-cancer activity that occurs in the Chinese medicinal plant Isodon rubescens and some related species. While the bioactivity of oridonin has been well studied, the extent of natural variation in the production of this compound is poorly known. This study characterizes natural variation in oridonin production in order to guide selection of populations of Isodon with highest oridonin yield. Different populations of I. rubescens and related species were collected in China, and their offspring were grown in a greenhouse. Samples were examined for oridonin content, genotyped using 11 microsatellites, and representatives were sequenced for three phylogenetic markers (ITS, rps16, trnL-trnF). Oridonin production was mapped on a molecular phylogeny of the genus Isodon using samples from each population as well as previously published Genbank sequences. Oridonin has been reported in 12 out of 74 species of Isodon examined for diterpenoids, and the phylogeny indicates that oridonin production has arisen at least three times in the genus. Oridonin production was surprisingly consistent between wild-collected parents and greenhouse-grown offspring, despite evidence of gene flow between oridonin-producing and non-producing populations of Isodon. Additionally, microsatellite genetic distance between individuals was significantly correlated with chemical distance in both parents and offspring. Neither heritability nor correlation with genetic distance were significant when the comparison was restricted to only populations of I. rubescens, but this result should be corroborated using additional samples. Based on these results, future screening of Isodon populations for oridonin yield should initially prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species, such as I. rubescens. Of the samples examined here, I. rubescens or I. japonicus from Henan province would provide the best source of oridonin.     

Mycorrhiza influences plant community structure in succession on spoil banks

The experiment simulated a plant succession stage when perennial grasses (e.g. Calamagrostis epigejos) invade communities of annuals with different mycotrophy (e.g. Atriplex sagittata and Tripleurospermum inodorum) on coalmine spoil banks. Communities of these three model species were planted in 30 l microcosms either in the presence of pre-established mycelium network of three arbuscular mycorrhizal fungi (AMF) species (individually and in a mixture) or without AMF. Different AMF species had significantly different effects on individual plant species’ growth, which resulted in changes in plant community structure. While in the no-AMF treatment the non-mycotrophic plant species A. sagittata contributed nearly 70% to the total plant biomass, in the presence of the 3 AMF mixture the contribution of this species was only about 10%. Different effects of AMF on tiller formation by C. epigejos suggest that some AMF could have greater potential to promote the replacement of annuals by perennial grasses than others. It can be concluded that not merely the presence but also the identity of AMF present on spoil banks can affect the coexistence of plant dominants, the community structure and the progress of plant succession.